The work of our team provides a valuable reference and experimental basis for future iGEM projects in many ways. Here is a detailed explanation of how each section contributes to future iGEM projects:
Truncated IFS enhances genistein production
In previous studies, it was found that the N-terminal hydrophobic structure of IFS proteins may lead to protein aggregation, which significantly affects their activity. Therefore, we truncated the N-terminal 21 amino acids of LjIFS to enhance its activity. This modification enhances the expression efficiency of the enzyme and increases the yield of genistein. Future iGEM teams can directly reference this approach to improve expression efficiency by tuning the N-terminal region of the enzyme, especially those working on plant metabolic engineering or natural product synthesis.
Figure 1.Yield of genistein in truncated LjIFS.
The OmpAL tag enhances CPR
In previous experiments, it was found that CPR is more difficult to functionally in E. coli, which may be due to the fact that its hydrophobic Nitrogen (N)-terminal tail functions as a membrane-anchored region in plant cells, while the lack of endoplasmic reticulum in Escherichia coli leads to the probability of reducing the solubility and functionality of these N-terminals. OmpAL can improve the efficiency of the conversion of naringenin to genistein. Overall, the addition of the OmpAL sequence can effectively improve the function of CPR after the results of this trial. For teams looking to express plant-derived membrane anchored proteins in bacteria, this experiment provides them with an effective strategy to improve protein solubility and functionality.
Figure 2. Comparison of genistein production before and after fusion of OmpAL tag.
The light-controlled induced suicide system ensures biosafety
To prevent genetic leakage, we have built a light-controlled suicide induction system to kill the live bacteria in the mask, and because most of the genistein is present in the bacteria, this can also release the genistein. We have verified the feasibility of a light-controlled suicide-induced suicide system by using a microplate reader to measure fluorescence values (excitation wavelength 584 nm, emission wavelength 607 nm) and OD600 values using a microplate reader, and to calculate a standardized fluorescence ratio (Fluorescence/OD600). Future teams, especially those working on environmental engineering or biologics, can learn from this approach to ensure the biosafety of their projects.
Figure 3 The genetic circuit diagram of the light-induced suicide system.
Adding new document to an existing Part:BBa_K4320012
According to the requirements of the bronze standard, we have added new document on the basis of the original existing Part. There is no experimental data on the existing Part page(https://parts.igem.org/Part:BBa_K4320012). We conducted both intracellular and extracellular validation experiments (Figure 4) and found that acsAB can promote the synthesis of intracellular cellulose. Future iGEM teams can use the results of our experiments to further investigate the cellulose synthesis pathway and apply the data in the field of industrial production or renewable resources.
Figure 4. Cellulose yield of recombinant strains. A Intracellular sample results. B Extracellular sample results.
Figure 5. We create new parts
Inclusivity
We have made the inclusive children's books, posters, card game rules and other related materials public and uploaded. These contents also provide a valuable reference for future iGEM teams. If future teams plan to develop similar projects, our results will provide them with inspiration and direction, helping them to better understand and deal with related challenges during the implementation of the project. We hope that these shared resources can support the research and practice of more teams, thereby jointly promoting the development of this field.
Conclusion
Our team’s work provides valuable contributions to future iGEM teams by enhancing enzyme efficiency, improving protein solubility, and ensuring biosafety. Key innovations include truncating the N-terminal region of LjIFS to boost genistein production, using an OmpAL tag to enhance CPR solubility in E. coli, and developing a light-controlled suicide system for biosafety. Additionally, we validated the intracellular cellulose synthesis capabilities of the acsAB part, offering data that can be applied in industrial and environmental contexts. Our inclusive outreach materials further support future teams in community engagement efforts.
